猪2型圆环病毒ORF2基因在塞姆利基森林病毒RNA复制子衍生的新型真核表达载体中的表达  

Expression of ORF2 Gene of Type 2 Porcine Circovirus in a Modified Eukaryotic Expression Vector Derived from RNA Replicon of Semliki Forest Virus

作　　者：罗玉子[1] 仇华吉[1] 刘建华[1] 韩凌霞[1] 李娜[1] 张守发[2] 童光志[1] 

机构地区：[1]中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室,哈尔滨150001 [2]延边大学农学院,龙井133400

出　　处：《生物工程学报》2004年第6期948-952,共5页Chinese Journal of Biotechnology

基　　金：黑龙江省科技攻关项目 (No .GA0 2B5 0 1)~~

摘　　要：猪 2型圆环病毒 (porcinecircovirus 2 ,PCV2 )是断乳仔猪多系统衰竭综合征 (postweaningmultisystemicwastingsyndrome,PMWS)的原发性病原。PCV2的ORF2编码病毒唯一的结构蛋白Cap。根据GenBank中公布的PCV2JXL株的序列设计一对引物 ,应用PCR方法从该毒株感染的PK 15细胞中扩增出完整的ORF2基因 ,将此基因克隆于本实验室此前构建的塞姆利基森林病毒 (SemlikiForestvirus,SFV)RNA复制子衍生的新型真核表达载体pSFV1CS中的BamHⅠ位点 ,获得重组质粒pSFV1CS Cap。用pSFV1CS Cap分别转染BHK 2 1细胞和 2 93T细胞 ,经间接免疫荧光试验检测表明 ,PCV2ORF2基因在转染细胞中得到表达。小鼠接种试验表明 。The ORF2 gene of postweaning multisystemic wasting syndrome (PMWS)-associated porcine circovirus 2 (PCV2) encodes the only structural protein, Cap.The ORF2 gene was amplified by PCR from the genomic DNA of PK-15 cell infected with PCV-2 JXL strain, then cloned into the unique BamHⅠ site of pSFV1CS, a eukaryotic expression vector derived from RNA replicon of Semliki Forest virus, creating a recombinant plasmid designated as pSFV1CS-Cap. The resultant plasmid was then transfected into BHK-21 and 293T cells. The results showed that the Cap gene was expressed efficiently in pSFV1CS-Cap-transfected cells, as demonstrated by indirect immunofluorescence assay (IFA).BALB/c mice inoculated with 10 or 100 μg of pSFV1CS-Cap developed anti-Cap protein specific antibodies, as indicated by IFA as well as indirect ELISA based on baculovirus-produced recombinant Cap protein, indicating that pSFV1CS-Cap can induce specific immune response in mice. The recombinant plasmid expressing PCV2 ORF2 gene can be used as a potential RNA vaccine to prevent PCV2 infections.

关 键 词：断乳仔猪多系统衰竭综合征 猪2型圆环病毒 塞姆利基森林病毒 RNA复制子 

分 类 号：S852.65[农业科学—基础兽医学]